Renu Geetha Baia,
Kasturi Muthoosamya,
Fiona Natalia Shiptonb,
Alagarsamy Pandikumarc,
Perumal Rameshkumarc,
Nay Ming Huangc and
Sivakumar Manickam*a
aManufacturing and Industrial Processes Research Division, Faculty of Engineering, University of Nottingham Malaysia Campus, 43500 Semenyih, Selangor, Malaysia. E-mail: Sivakumar.Manickam@nottingham.edu.my
bSchool of Pharmacy, Faculty of Science, University of Nottingham Malaysia Campus, 43500 Semenyih, Selangor, Malaysia
cLow Dimensional Materials Research Centre, Department of Physics, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia
First published on 29th March 2016
Cancer nanotechnology encourages cutting edge research utilizing nanomaterials for the diagnosis, therapy and prevention of cancer. Recognition of cancer-related biomarkers in the body has made early detection possible and thus, paves the way towards devising methods to control it from progressing to advanced stages. Hydrogen peroxide (H2O2) is a critical biomolecule, which plays an important dual role in cancer progression. Herein, we have developed a sensitive method for the detection of H2O2 utilizing a reduced graphene oxide–silver (RGO–Ag) nanocomposite. This RGO–Ag nanocomposite was prepared using a green and facile one-step synthesis approach utilizing the extract of a medicinal mushroom, Ganoderma lucidum. The higher content of polysaccharides in this extract makes it a potent reducing agent for the combined reduction of GO and AgNO3 to produce the RGO–Ag nanocomposite. The properties of the RGO–Ag obtained were characterized by UV-Vis spectroscopy, SEM, TEM, XRD, FT-IR and XPS techniques. The RGO–Ag modified electrode showed good electrocatalytic activity towards H2O2 when compared to other modified electrodes. Furthermore, it showed an LOD of 136 nM, which was determined using the LSV technique. The amperometric i–t curve displayed two different linear ranges of 1–100 μM and 100–1100 μM with an LOD of 3 and 56 nM, respectively. This excellent electrochemical performance towards H2O2 detection could contribute to advances in current cancer diagnosis. The RGO–Ag nanocomposite was also explored as a potential antibacterial agent. Owing to its synergistic effects, RGO–Ag showed a comparable antibacterial activity to the standard antibiotic, chloramphenicol. The combined antibacterial effects and sensing potential make this RGO–Ag nanocomposite a promising candidate for future health care.
The antibacterial activity of silver is well documented; the properties of AgNPs vary based on their size and distribution. The antibacterial effects of AgNPs arise due to the following: (1) the action of the AgNPs themselves, (2) silver ions released from AgNPs and (3) reactive oxygen species (ROS) such as superoxide, hydrogen peroxide or hydroxyl radicals generated from the AgNPs.15–17 In addition, the high surface area of AgNPs facilitates the contact with microorganisms to penetrate through the cell membrane. However, the size associated surface energy promotes the agglomeration of AgNPs, which can result in the loss of their antibacterial properties. Owing to its high surface area, graphene serves as an excellent supporting material for the in situ growth of AgNPs.18 Moreover, graphene and its derivatives also possess antimicrobial potential, which is triggered via either membrane stress or superoxide anion independent oxidation.19–21 The synergistic antibacterial effects of reduced graphene oxide (RGO) and AgNPs in the graphene–silver nanocomposite (RGO–Ag) makes it an ideal antibacterial agent.22–24 Furthermore, when compared to its individual counterparts, in the case of the RGO–Ag nanocomposite, the high surface area of RGO maximizes the contact of AgNPs with bacterial cells and consequently improves the bactericidal activity.25 The RGO–Ag hybrid material has also been comprehensively examined for its electrocatalytic activity and it has been reported as an appropriate platform for the detection of chemicals such as dopamine, peroxides, glucose, ammonia and chloride.26–30
Hydrogen peroxide (H2O2) is a key biomolecule involved in diverse biological responses. It plays a vital dual role in carcinogenesis. Increased levels of H2O2 are reported to be associated with DNA damage, gene instability, apoptosis resistance, increased angiogenesis, metastasis etc. On the contrary, selective anti-cancer effects have also been reported with increased cellular levels of H2O2. Being a significant biomarker involved in numerous biological signaling pathways, a balance in the H2O2 levels is achieved by the antioxidant enzymes such as catalase, glutathione peroxidase and thioredoxin peroxidase. Thus, the detection of fluctuations in the cellular levels of H2O2 is highly imperative in different disease diagnoses.31–34
Green synthesis using phytochemicals is an economic and valuable alternative for the large-scale production of nanomaterials, which are environmentally benign, yet chemically complex. Plant extracts perform as efficient reducing and capping agents in nanoparticle synthesis. When compared to conventional physical and chemical synthesis approaches, bioreduction through phytochemicals leads to the synthesis of highly stable, well dispersed and eco-friendly nanomaterials, which are compatible for bio-applications due to the toxic-free synthesis approaches.35 Moreover, green synthesis has the benefits of using non-hazardous materials, the production of non-hazardous waste, less processing effort and ease of reproducibility.36 Recently, many investigations have reported on biogenic synthesis approaches utilizing plant and fungal extracts for the synthesis of graphene–silver nanocomposites and the evaluation of their antimicrobial potential.37–39
In this investigation, a RGO–Ag nanocomposite was synthesized using a green route using Ganoderma lucidum (G.l.) extract as a strong reducing agent.40 The rich content of polysaccharide in this mushroom extract assists the combined reduction of both graphene oxide (GO) and silver nitrate to produce the RGO–Ag nanocomposite. The prepared RGO–Ag was characterized to understand the morphological, structural, physical and chemical properties using UV-Vis spectroscopy, XRD, XPS, FT-IR, TGA, SEM, HRTEM and EDAX analysis. The application of the RGO–Ag nanocomposite as an antibacterial material was investigated on 4 different bacteria by adopting qualitative and quantitative approaches. In addition, the electrocatalytic potential of the RGO–Ag nanocomposite was exploited to create an electrochemical sensor for the detection of H2O2. The results validate the successful use of the RGO–Ag nanocomposite for both antibacterial and electrochemical sensing applications, which could be produced in a cost-effective, non-toxic and simple one-step method.
For the disc diffusion analysis, the RGO, AgNPs and RGO–Ag samples were compared with chloramphenicol, a standard broad spectrum antibiotic used as the positive control. Plain paper discs were used as the negative control. Autoclaved RGO, AgNPs and RGO–Ag samples and the antibiotic were loaded onto the paper discs by a drop drying method to obtain the required quantity of 50 μg per disc. The bacteria were grown in MH broth and the optical density was adjusted to 0.5 (measured using a UV-Vis spectrometer) before streaking onto agar plates. The prepared discs were placed evenly onto the surface of the agar and labelled. The agar plates were kept inverted in an incubator at 37 °C for 24 h. The diameter of the zone of inhibition was then measured and used to evaluate the antimicrobial action.
To analyze the minimum inhibitory concentration (MIC) of RGO–Ag, the broth dilution technique was used. Colour controls were kept to avoid any misinterpretation due to the colour of the RGO, AgNPs and RGO–Ag samples. The broth dilution test was conducted in a 96-well plate by preparing different concentrations of RGO, AgNPs, RGO–Ag and chloramphenicol in MH broth. The bacteria were grown in MH broth and the optical density was adjusted to 0.5 before further use. The RGO, AgNPs, RGO–Ag and chloramphenicol were added to a 96-well plate and 10 μL of the bacterial suspension was introduced to each well and incubated for another 24 h. The plates were read using a microplate reader to analyze the MIC of RGO, AgNPs, RGO–Ag and chloramphenicol for each bacterium at 600 nm. The lowest concentration that completely inhibited the bacterial growth was recorded as the MIC.
To determine the minimum bactericidal concentration (MBC), 10 μL of RGO, AgNPs, RGO–Ag and chloramphenicol from each well that showed no bacterial growth was applied to the MH agar plate and incubated overnight before checking for bacterial growth. The MBC value was evaluated by calculating the lowest concentration of sample that was required to kill the bacteria.
To visualize the bactericidal effect of the RGO–Ag nanocomposite, the morphology of the bacterial cells was examined using SEM. For comparison, images of RGO-Ag treated and untreated bacteria were captured. Bacterial cells for SEM were prepared using a previously reported protocol43 by fixing the bacterial cells on the SEM stubs with double-sided carbon tape.
RGO exhibited a peak at 260 nm, which is a red-shift from the characteristic peak of GO at 230–240 nm (Fig. 1a). The AgNPs exhibited a strong broad peak at 470 nm, which corresponds to the surface plasmon resonance band.44 RGO–Ag showed an obvious blue-shift with a sharp peak positioned at 410 nm corresponding to the AgNPs, where the RGO peak was not obviously visible possibly due to the high intense peak arising from the deposition of AgNPs on the RGO sheets. The blue-shift of the plasmonic nanomaterials is associated with the structure, size as well as the properties of the substrate. Here, the observed peak shift could be either due to a decrease in the size of the AgNPs or due to the presence of RGO, the matrix on which the AgNPs were deposited.45,46 When the RGO–Ag reaction was continued for 24 h, the Ag peak showed a slight red-shift with a broader peak and a shoulder appearing around 565 nm, which could be due to the agglomeration of the AgNPs.
The morphological analysis of the synthesized RGO, AgNPs and RGO–Ag were carried out by SEM, which exhibited few layers of separated and transparent RGO sheets as shown in ESI Fig. S1a.† The AgNPs exhibited agglomeration in Fig. S1b,† whereas in Fig. S1c† the RGO–Ag showed uniform sized AgNPs (<15 nm) deposited on the RGO sheets with little agglomeration, which could be due to the air drying step. Fig. S1d† confirmed the uniform deposition of the different sized AgNPs on the few-layered RGO surfaces after 24 h of reaction, which supported the UV-Vis data analysis (Fig. 1d).
HRTEM analysis was performed to gain a better understanding of the morphological nature of the prepared RGO–Ag nanocomposites. Based on the HRTEM images, AgNPs with an average diameter of 6.02 ± 0.31 nm were found to be uniformly distributed on the RGO as displayed in Fig. 2a and b.
Fig. 2 HRTEM images of (a) RGO–Ag, (b) lattice resolved image of the AgNPs in the RGO–Ag nanocomposite and (c) the number based size distribution plot of the RGO–Ag nanocomposite. |
The HRTEM image shows the lattice fringes of the AgNPs with an interlayer spacing of 0.26 nm between the boundaries, which correspond to the (111) plane and confirms the crystalline nature of the AgNPs. In the case of RGO–Ag, the deposition of the AgNPs on the surface of RGO effectively prevents the restacking possibility of the RGO sheets, maintaining the few layer structure of the RGO–Ag nanocomposite. Size distribution analysis was performed by HRTEM analysis based on the number of AgNPs present on the RGO surface as shown in Fig. 2c. The particle size distribution of the RGO–Ag nanocomposite was executed by counting more than 2200 nanoparticles with Image J software considering different HRTEM images. The size study data was fitted with the Gaussian distribution curve and the calculated average size was 6.02 ± 0.31 nm. The size distribution of the AgNPs was in a range of 1–15 nm based on the HRTEM images. The interaction of the AgNPs with RGO could be attributed to the chemical affinity of AgNPs to the remaining oxygen functional groups in the RGO or to the influence of the reducing polysaccharides.38 However, the exact mechanism behind anchoring the AgNPs onto the RGO surface in the RGO–Ag nanocomposite is yet to be unveiled.
Elemental analysis was carried out using EDAX, which gives the weight and atomic percentage of the elements present in RGO, AgNPs and the RGO–Ag nanocomposites. As shown in Fig. 3a, the AgNPs show a small percentage of silicon and oxygen content, which is from the silica glass used to prepare the AgNPs for analysis. Since RGO was synthesized via the reduction of GO, the presence of oxygen was still noticeable, possibly due to the remaining oxygen functional groups in the air-dried RGO–Ag. Analysis of the AgNPs showed stronger Ag peaks, representing 72.24 weight% of Ag content with a small weight% of carbon (9.53), which could originate from the G.l. extract. In the case of RGO–Ag, the weight% of Ag and carbon content were 67.85 and 18.43, respectively, which indicates that AgNPs have been deposited on the surface of RGO as seen from the HRTEM images (Fig. 2).
Fig. 3 Characterization of RGO, AgNPs and the RGO–Ag nanocomposites (a) EDAX analysis, (b) XRD analysis of the synthesized, (c) TGA and (d) FTIR analysis. |
Fig. 3b demonstrates the XRD analysis of RGO, AgNPs and the RGO–Ag nanocomposites. RGO showed a characteristic peak at an angle of 22° for the (002) crystal plane. According to the JCPDS files 04-0783 and 84-0713, the observed diffraction peaks at 2θ values of 38°, 46° and 64° correspond to the (111), (200) and (220) crystal planes of the AgNPs.47 The diffraction pattern of RGO–Ag showed similar peaks, confirming the presence of AgNPs. In addition, RGO–Ag exhibited a peak at 22° and 28° due to the (002) crystalline planes of RGO.48 The peak observed in RGO at 32°, which is usually present in most of the phytochemical mediated green synthesis approaches is considered to be due to the crystallization of bioorganic contents from the mushroom extract deposited on the surface of AgNPs.49
The thermal stability of RGO, AgNPs and RGO–Ag were evaluated using TGA, which determines the percentage of weight loss upon an increase in temperature (Fig. 3c). RGO showed a 19% weight loss upon heating to 125 °C owing to the removal of water in RGO after the reduction of GO. From 125 to 840 °C, RGO followed a gradual weight loss of <2%, which demonstrated the removal of the remaining functional groups such as carboxylic, anhydride or lactone. Finally, a sudden decrease of 38% occurred from 840 to 1000 °C, possibly due to the bulk pyrolysis of the carbon skeleton.50
Thermal stability analysis of the AgNPs resulted in three steps. Until 300 °C, the AgNPs exhibited negligible weight loss due to the removal of water. From 300 to 500 °C a sharp decrease in the weight (70%) was observed, which could be attributed to the thermal degradation caused by an amorphous to crystalline transition of the AgNPs. After 500 °C, there was a gradual weight loss of <10% demonstrating the stability of the AgNPs in this temperature range. The TGA spectrum of the RGO–Ag nanocomposite confirmed a stable profile with a minimum weight loss of less than 10% until 445 °C. From 445 to 650 °C, the RGO–Ag was subjected to a 40% weight loss; thereafter it showed thermal stability with negligible weight loss (below 5%). Among all these materials, RGO–Ag showed the highest thermal stability followed by RGO and then the AgNPs.
FTIR analysis evaluated the chemical functionalities present in RGO, AgNPs, the RGO–Ag nanocomposite and G.l. extract as shown in Fig. 3d. Due to the water-mediated green synthesis process, two peaks were observed at 3400 cm−1 and at 1400 cm−1 corresponding to –OH stretching vibrations. When compared to the G.l. extract and GO, the intensity of these peaks was smaller in other two spectra i.e. RGO and the RGO–Ag nanocomposite. The involvement of G.l. extract as a green reducing agent during the reduction could be the source of these hydroxyl groups in RGO and RGO–Ag. The amorphous nature of RGO and RGO–Ag identified using XRD also supports this finding. In the G.l. extract and GO, the peak observed in the range of 1600 cm−1 corresponds to the CO stretch of carbonyls or carboxylic acids. Since RGO is synthesized by the reduction of GO, similar peaks were also observed due to the residues of oxy-carboxyl functional groups. Another intense peak observed in the G.l. extract and GO at 1050 cm−1 corresponds to the CO stretching vibration and the intensity of this peak was significantly reduced after the reduction of GO.
XPS analysis of RGO revealed the presence of a predominant C 1s peak at 284.5 eV and O 1s peak at 532 eV (Fig. 4a). The C 1s indicated the presence of various functional groups in the RGO film such as CC at 284.2 eV, C–OH at 285.1 eV, C–O at 286.2 eV, CO at 287.4 eV and COOH at 288 eV. These peaks exhibited the presence of sp2 and sp3 carbons in the RGO.51–53 The XPS of metallic silver is usually represented in two prominent peaks at 367.9 eV and at 373.9 eV with spin energy of 6 eV. These AgNPs exhibited a shift in the peaks with same spin energy separation, which indicated the generation of zero-valent AgNPs.54 Fig. 4c shows the XPS data of RGO–Ag, where the spectrum of RGO indicated a shift in the bonds similar to native RGO with C 1s at 284.4 eV and O 1s at 532.2 eV and five different bonds: C–C at 284.4 eV, C–OH at 284.9 eV, C–O at 286.1 eV, CO at 287.7 eV and COOH at 288.7 eV. As shown in Fig. 4c, the Ag 3d spectrum expressed two peaks with the binding energies of the 3d5/2 and 3d3/2 electrons for AgNPs at 367.766 and 73.766 eV, respectively with a spin energy difference of 4 eV.55
The change in spin energy from 6 eV to 4 eV could be attributed to the attachment of AgNPs on the surface of the RGO sheets.
These plates were incubated at 37 °C for 24 h to obtain the bacterial clearance zone as shown in Fig. 5. The clearance zone represents the inhibition of bacterial growth, which is due to the antibacterial activity of the material deposited on the paper discs. Fig. 5A and C represented the comparable antibacterial activity of RGO–Ag towards S. aureus and E. coli, respectively with the standard antimicrobial agent, chloramphenicol. When compared to chloramphenicol, S. aureus exhibited 48.14% of antibacterial activity and E. coli exhibited 48.27%, whereas in Fig. 5B and D the B. cereus and P. aeruginosa expressed lesser antibacterial activity of 25.89% and 11.48%, respectively.
For the disc diffusion analysis of the RGO–Ag nanocomposite, S. aureus exhibited a 2-fold antibacterial activity when compared to the other Gram-positive bacteria i.e., B. cereus. By the same token, the antibacterial action of the RGO–Ag nanocomposite in E. coli was 4 times higher when compared to P. aeruginosa. These results supported the bacterial species-specific antibacterial properties exhibited by the RGO–Ag nanocomposite. The clearance zone diameter is shown in Table 1.
Bacteria | RGO | AgNPs | RGO–Ag | Chloramphenicol | Plain disc |
---|---|---|---|---|---|
Staphylococcus aureus | — | 9.17 ± 0.29 | 13.00 ± 0 | 27.00 ± 0 | — |
Bacillus cereus | — | 1.17 ± 0.29 | 3.33 ± 0.29 | 29.00 ± 0 | — |
Escherichia coli | — | 6.33 ± 0.29 | 14.00 ± 0 | 29.00 ± 0 | — |
To gain a more detailed understanding of antibacterial properties of RGO–Ag, the MIC and MBC values were evaluated (ESI Fig. S2†). For the MIC calculation, different dilutions of RGO, AgNPs, RGO–Ag and the standard chloramphenicol were prepared and a broth dilution test was conducted in 96-well plates. Each 96-well plate was inoculated with different bacteria (10 μL) and tests were carried out in triplicate with five different concentrations of the RGO, AgNPs, RGO–Ag and 7 concentrations of chloramphenicol (Fig. S2a†). The concentration of RGO, AgNPs, RGO–Ag and chloramphenicol that killed 99.9% of the bacteria was considered as the MBC value. To analyze the MBC, the 24 h broth dilution test samples were utilized. 10 μL of dilutions (which showed no bacterial growth) of RGO, AgNPs, RGO–Ag and chloramphenicol, were taken from the selected wells, spotted onto new agar plates and incubated for 24 h in an incubator at 37 °C (Fig. S2b†). The MBC values of all 4 bacterial strains were calculated by checking the presence of colonies after 24 h.56 The MIC and MBC values of RGO, AgNPs, RGO–Ag and chloramphenicol are displayed in Table 2.
Material | RGO | AgNPs | RGO–Ag | Chloramphenicol | ||||
---|---|---|---|---|---|---|---|---|
Bacteria | MBC | MIC | MBC | MIC | MBC | MIC | MBC | MIC |
Staphylococcus aureus | >1000 | 500 | >1000 | >1000 | 500 | <62.5 | 250 | <7.8125 |
Escherichia coli | >1000 | 1000 | 1000 | 250 | ≥1000 | <62.5 | 125 | <7.8125 |
Bacillus cereus | >1000 | >1000 | 500 | 250 | >1000 | 1000 | 500 | <7.8125 |
Pseudomonas aeruginosa | >1000 | >1000 | <500 | 500 | 1000 | 125 | >1000 | <125 |
AgNPs mediated antibacterial effects are often explained in association with silver ion induced ROS generation inside cells, leading to irreversible changes in the genetic structure of the microbes leading to cell cycle arrest and death. RGO based antibacterial effects are linked with membrane and oxidative stress. Despite these proposed mechanisms, the influencing factors and the actual mechanism behind the antibacterial properties of silver and graphene based nanomaterials are still unclear. From the observations of our qualitative and quantitative investigations, the antibacterial properties of RGO, AgNPs and the RGO–Ag nanocomposite were varied for each bacterium. However, the RGO–Ag nanocomposites exhibited supreme antibacterial effect in the disc diffusion analysis among all the tested bacteria except B. cereus. In the case of B. cereus, the AgNPs showed a better antibacterial potential than the RGO–Ag nanocomposites.
To investigate the bactericidal effects of the RGO–Ag nanocomposite, SEM images of E. coli and S. aureus were evaluated (as a model for Gram-positive and Gram-negative bacteria) with and without the RGO–Ag treatment. As shown in Fig. 6a and c, without RGO–Ag treatment, both E. coli and S. aureus were grown healthy under normal culturing conditions, whereas after treatment with RGO–Ag for 24 h, the E. coli and S. aureus images (Fig. 6b and d) showed the presence of very few bacterial cells. The cells were found to be wrinkled, damaged or completely covered by the RGO–Ag. In the case of E. coli, the cells were fragmented into smaller parts (marked in Fig. 6b), which indicated complete cell damage or cell membrane disruption. No healthy bacterial cells were observed after the treatment. This clearly indicated the cell integrity disruption effect of the RGO–Ag nanocomposites in Gram-negative bacteria. Similarly, in the case of S. aureus, very few cells were observed after treatment with RGO–Ag. Though the morphology of S. aureus remains unaltered, the bacterial cells were completely covered by the RGO–Ag nanocomposite (marked in Fig. 6d), which could hinder the growth of the bacteria. Furthermore, in a similar study using the GO–Ag nanocomposite, E. coli and S. aureus exhibited completely distinctive antibacterial effects. E. coli resulted in the disruption of the bacterial cell membrane, but in the case of S. aureus the antibacterial effect inhibited cell division, with little cell wall damage.
Interaction with nanoparticles creates perforations in the cell membrane of bacteria and thus, changes in its permeability. These perforations enable the nanoparticles to enter the bacterial cell and interfere with the growth signalling pathways and thereby alter the cell viability and division.57 Here, the rod shaped Gram-negative E. coli cells showed complete damage to the integrity of the cell membrane and were found to be smaller pieces after treatment. At the same time, the Gram-positive S. aureus cells showed no alteration from their original spherical morphology, but the cells were found to be completely covered with the RGO–Ag nanocomposite. This could be attributed to the difference in cell wall structure and chemical composition of the Gram-positive and Gram-negative bacteria. When compared to the delicate thin peptidoglycan cell membrane in Gram-negative bacterial cells, the Gram-positive bacteria possess a cell wall comprised of multiple layers of peptidoglycan, which provides better cell membrane integrity and thus, prevents the cell disruption.58 However, the disc diffusion analysis of the RGO–Ag nanocomposites exhibited only 11.48% activity towards P. aeruginosa, whereas it showed 48.14% activity towards S. aureus, when compared to chloramphenicol. Similarly, in the broth dilution test, S. aureus exhibited an MBC of 500 μg mL−1 and an MIC of <62.5 μg mL−1, whereas P. aeruginosa exhibited an MBC and an MIC of 1000 and 125 μg mL−1, respectively, which is twice the concentration of RGO–Ag needed for S. aureus. Considering the bacterial cell structural composition, P. aeruginosa belongs to the Gram-negative family, a more susceptible strain to disruption in terms of the nanoparticle mediated cell wall disruption. In contrary, S. aureus (Gram-positive strain) was more affected by the bactericidal action of the RGO–Ag nanocomposite. This difference in response for each bacterium directs the complex molecular mechanisms involved in the antibacterial effect. Besides, these results support the hypothesis of the species-specific antibacterial response of the RGO–Ag nanocomposite where the bacterial species “aureus” showed better resistance compared to the other species “aeruginosa”. However, when RGO–Ag showed minimal antibacterial response, another similar reported study utilizing GO–Ag nanocomposite resulted in a remarkable bactericidal response for P. aeruginosa, which points out the aspect of material composition based antibacterial action.59 Herein, among the three nanomaterials, RGO was observed to be the most biocompatible to bacterial cells when compared to AgNPs and the RGO–Ag nanocomposites. A molecular or proteomic level study could be useful towards understanding the exact mechanism behind the bactericidal activity of this RGO–Ag nanocomposite.
Prior to electrode modification, the CV responses of the bare GC were obtained with 1 mM K3[Fe(CN)6] in 0.1 M KCl at a scan rate of 50 mV s−1 to ensure the reliability of the electrode. The bare GC electrode displayed a reversible voltammetric characteristic for the [Fe(CN)6]3−/4− couple with a peak-to-peak separation of 70 mV at a scan rate of 50 mV s−1. To optimize the electrocatalytic activity, the modified electrode was fabricated with different dilutions of RGO–Ag and the electrocatalytic reduction of H2O2 was analyzed in the presence of 0.1 mM H2O2. The best catalytic activity towards H2O2 was observed with a 4 times dilution of the synthesized RGO–Ag with a high catalytic reduction peak (ESI Fig. S3†). The CV analysis was repeated with different GC electrodes to confirm the reproducibility and reliability of the results obtained.
Following the optimization of the best dilution of RGO–Ag, the cyclic voltammograms were recorded for the bare GC and GC modified with RGO–Ag, RGO and AgNPs in the presence of 1 mM H2O2 in 0.1 M PBS (pH 7.2). As shown in Fig. 7, the GC/RGO–Ag modified electrode displayed a higher catalytic response with a peak potential of −0.519 V towards the reduction of H2O2 while other modified electrodes exhibited lower voltammetric responses in the same potential window. Moreover, in the absence of H2O2, the RGO–Ag nanocomposite modified electrode did not display any peak currents, which confirm the reliability of electrochemical reduction.
The effect of different scan rates was obtained for the GC/RGO–Ag modified electrode upon the addition of 10 μM H2O2 in 0.1 M PBS (ESI Fig. S4†). The scan rate differed from 10 mV s−1 to 100 mV s−1 on successive readings. The CV responses were recorded to analyze the linear relation of current with respect to change in the scan rate.
The linear sweep voltammogram (LSV) obtained for the GC/RGO–Ag modified electrode upon the addition of 10 μM H2O2 in 0.1 M PBS is presented in Fig. 8a. This LSV result reveals the change in the peak current in accordance with the increase in the concentration of H2O2. The inset picture gives the calibration plot for the peak current against H2O2 concentration, where it displayed a linear relationship. The calibration plot shows a correlation coefficient (R2) of 0.9837 using the regression equation of I (μA) = 0.038[H2O2] μM + 5.998. The limit of detection was calculated as 136 nM with the signal-to-noise ratio (S/N = 3) by substituting the blank standard deviation (σ) in the 3σ/m criterion.
The electrochemical detection of H2O2 was also performed via amperometric analysis using this RGO–Ag based enzymeless sensor. The amperometric i–t curve responses were recorded for successive additions of H2O2 at a regular time interval of 60 s in 0.1 M PBS (Fig. 8b). In order to eliminate interference from any electroactive chemicals present in the solution, the potential applied for amperometric analysis was −0.51 V. For the successive addition of H2O2, an enhancement in the current response was recorded. The current versus H2O2 concentration curve in the range of 1–1100 μM is displayed in Fig. 8b (inset). The current proportionally increased with an increase in the concentration of H2O2. In addition, two different linear segments were observed in the calibration plot of current versus H2O2 concentration. The first linear segment (I (μA) = 0.003[H2O2] μM + 1.653) corresponds to H2O2 concentration from 1–100 μM, while the second linear segment (I (μA) = 0.0078[H2O2] μM + 1.266) corresponds to H2O2 concentration from 100–1100 μM. A decrease in the sensitivity at higher concentrations of H2O2 is due to the kinetic limitation of the modified electrode. The LOD for H2O2 in the lower and higher concentration range was found to be 3 and 56 nM, respectively.
Non-specific binding and insensitivity are the major problems encountered when developing a biosensor. To investigate the selectivity and specificity of the sensor electrode towards H2O2, an interference study was performed using the RGO–Ag/GC electrode with interfering biomolecules such as glucose, dopamine, ascorbic acid, nitric acid and sodium chloride. As shown in Fig. 8c, after introducing H2O2, 100 times higher concentrations of the interfering chemicals were added to the electrochemical cell; but no signals were generated by the addition of these chemicals. This confirmed the sensitivity of the electrode towards H2O2 detection even in the presence of interfering chemical entities at higher concentrations. H2O2 was again spiked into the system after the introduction of the interference molecules. For each addition of H2O2, the modified electrode gave a corresponding catalytic response as a current enhancement. The selective identification and response to H2O2 confirmed the sensitivity and specificity of the RGO–Ag modified GC electrode.
Upon comparison the recent studies of similar graphene modified electrodes, as shown in Table 3, our RGO–Ag modified electrode exhibited an LOD of 0.136 μM using the LSV technique, which is comparable to the similar detection limit observed with other sensing studies reported in the literature. Following the amperometric analysis, RGO–Ag gave a clearly enhanced detection response with a LOD of 0.003 μM, which is the lowest detection limit reported for GC based enzymeless sensing of H2O2. This clearly defines the potential of the RGO–Ag nanocomposite as a competent sensing material.
Sensing electrode | Synthesis approach | Mode of detection | Detection limit (μM) | Linear range (μM) | Reference |
---|---|---|---|---|---|
a Cu – copper; CA – chronoamperometry; MWCNTs – multi-walled carbon nanotubes; MOF – metal organic framework; DNA – deoxyribonucleic acid; AgNCs – Ag nanoclusters; ITO – indium tin oxide; HRP – horseradish peroxidase; P-L-His – poly L-histidine; SWV – square wave voltammetry; LSV – linear sweep voltammetry. | |||||
Nafion/Cu | Electrochemical deposition | CA | 1.63 | 1–400 | 62 |
3D-RGO–Ag | Electrochemical reduction | CA | 1.6 | 100–9000 | 63 |
Gold/Nafion: polypyrrole/MWCNTs | Sonochemical synthesis | CA | 1.47 | 5–30 | 64 |
3D-CuMOF | Solvothermal method | CA | 1 | 1–900 | 65 |
DNA–AgNCs/graphene | Immobilization by π–π conjugation | CA | 3 | 15–23 | 66 |
AgNPs–RGO/ITO | Electrodeposition | CA | 5 | 0.1–100 | 67 |
AgNPs–RGO/ITO–HRP tagged antibody | In situ electrochemical deposition | CV | 214 | 25–500 | 68 |
CA | 5.3 | 25–1450 | |||
HRP/P-L-His–RGO | Layer-by-layer assembly | CA | 0.05 | 0.2–5000 | 69 |
GE–CNT–Nafion/AuPt NPs | Electrochemical deposition and immobilization | SWV | 0.17 | 0.5–100 | 70 |
RGO–Ag | Phytochemical synthesis | LSV | 0.136 | 10–1000 | This work |
CA (two linear responses) | 0.003 | 1–100 | |||
0.056 | 100–1100 |
Footnote |
† Electronic supplementary information (ESI) available: Information on the RGO–Ag nanocomposite regarding additional characterization data, antibacterial experiments and electrocatalytic responses. See DOI: 10.1039/c6ra02928k |
This journal is © The Royal Society of Chemistry 2016 |